Treatment of anaplasmosis in animals

ABSTRACT

A METHOD FOR THE TREATMENT OF ANAPLASMOSIS IN CATTLE, COMPRISING THE INTRAVENOUS OR ORAL ADMINISTRATION OF AN EFFECTIVE DOSAGE OF A-ETHOXYETHYLGLYOXAL DITHIOSEMICARBAZONE TO THE INFECTED ANIMAL.

United States Patent 3 592 915 TREATMENT OF ANAPIiASMOSIS 1N ANIMALSPaul Anthony Barrett, London, England, assignor to Burroughs Wellcome &Co. (U.S.A.) Inc., Tuckahoe,

No Drawing. Continuation-impart of application Ser. No. 388,717, Aug.10, 1964, now Patent No. 3,382,275, dated May 7, 1968. This applicationMar. 6, 1968, Ser. No. 710,789

Claims priority, application Great Britain, Aug. 10, 1963,

31,673/ 63 Int. Cl. A61k 27/00 U.S. Cl. 424323 8 Claims ABSTRACT OF THEDISCLOSURE A method for the treatment of anaplasmosis in cattle,comprising the intravenous or oral administration of an effective dosageof a-ethoxyethylglyoxal dithiosemicarbazone to the infected animal.

This invention relates to the methods of preventing and treatinganaplasmosis in cattle. The application is a continuation-in-part ofapplication No. 388,717 of Aug. 10, 1964, now U.S. Pat. 3,382,275 issuedMay 7, 1968.

In the specification of the above application there are described andclaimed substituted glyoxal dithiosemicarba- Zones, having activityagainst anaplasmosis in cattle. It has also been stated that some of thedisclaimed compounds of this class have been mentioned in theliterature, but no activity against anaplasmosis has been attributed tothem.

An object of the present invention is to provide a satisfactory methodof treating this disease with a compound which shows sufiicient highactivity without rendering other microbes present in the animalresistant to important antibiotics and antibactericides frequently usedin medical and veterinary practice.

It has now been found that a-ethoxyethylglyoxal dithiosemicarbazone,which is a member of the aforementioned group has outstanding activityin this respect and can advantageously be used in the prevention ortreat ment of anaplasmosis in cattle.

Anaplasmosis is a serious systemic disease of cattle which is prevalentin large areas of the Asian, African, American and Australasiancontinents and certain Southern areas of Europe which have amediterranean type of climate. The infecting organism Anaplasmamarginale attacks the red blood cells of the cattle characteristicallycausing anaemia, general debility, and fever of the animal, which oftenprove fatal.

The present invention, therefore, provides a method for the treatment ofanaplasmosis in cattle, comprising the intravenous or oraladministration of an effective dosage of a-ethoxyethylglyoxaldithiosemicarbazone of the infected animal. Usually a dosagerepresenting about 20 mg./kg. (orally) or 10 mg./kg. (intravenously) issufficient for this purpose; significant improvements can occasionallybe achieved with considerably lower dosages, such as about 5 mg./kg. oreven 2.5 mg./kg.

The natural incubation period for anaplasmosis is about to 50 days incattle. Thus the smallest animal likely to be treated would be aseven-weeks old calf which would weigh about to kg. or more. Treatingsuch an animal would require 200 to 500 mg. at a dose of about 5 to 10mg./kg., but adult beasts would, of course, require a much larger dose.

The treatment of anaplasmosis in cattle according to the presentinvention can also be used for the premunition of the animals. After anartificial infection of the animal with a suitable strain of A.marginale, the disease is treated with a-ethoxyethylglyoxaldithiosemicarbazone to prevent its further development. The animalremains "Ice a carrier of A. marginale and develops sufficientpremunition to resist subsequent field challenge. It has been found thatdosage of about 5 to 10 mg./ kg. intravenously administered after thedevelopment of parasites gives satisfactory results.

The present invention, in another aspect, provides a method ofpreventing anaplasmosis in cattle, comprising the repeatedintramuscular, subcutaneous or oral administration ofa-ethoxyethylglyoxal dithiosemicarbazone in a dosage of about 1 mg./kg.day to the animal for 10 to 30 days. In a further aspect, there isprovided a method of sterilising the infection with A. marginale incarrier cattle, comprising the oral administration of 0:-ethoxyethyldithiosemicarbazone at a dosage of about 1 mg./kg. day for upto 30 days. There are many animals 'which have survived the infection,but remain carriers and a source of further infection, and a treatmentwith this medicament can eliminate the danger in this respect.

aEthoxyethylglyoxal dithiosemicarbazone may be prepared by adaptation ofany of the methods known to be useful for converting compoundscontaining a ketone or aldehyde group into their thiosemicarbazonederivatives. Conveniently it is prepared by reacting in an acid mediumtwo molecular proportions of a thiosemicarbazide of formula NH NH.CS.NHwith a glyoxal of formula EtO.CH(Me).CO.CHO. The reaction may beeffected with heating in a solvent, for example ethanol or aqueousethanol, preferably in the presence of a trace of mineral acid, forexample hydrochloric acid, as a catalyst. The compound is in generalsparingly soluble and separates from the hot reaction mixture; afterfiltration and washing, and possibly re-crystallisation, it is obtainedin a pure state.

a-Ethoxyethylglyoxal dithiosemicarbazone may be presented inpharmaceutical formulations suitable for oral or parentheraladministration. For example, the oral preparations may be tablets,capsules, granules, powder, suspension, or solutions, which may containdiluents, binding agents, dispersing agents, surface-active agents,lubricating agents, coating materials, colouring agents, solvents,thickening agents, suspending agents, or other therapeuticallyacceptable additives, and these preparations may be presented inunit-dose form or multi-dose form, or as additives to feed-stuffs. Theinjectable form may be a nonaqueous solution for instance in dimethylsulphoxide, or suspension in a therapeutically acceptable liquid ormixture of liquids, which may contain bacteriostatic agents,antioxidants, buffers, solutes to render the solution isotonic with theblood, thickening agents, suspending agents, or other therapeuticallyacceptable additives. Such preparations are presented in unit-dose formssuch as ampoules or disposable injection devices, or in multidose formssuch as a bottle from which the appropriate doses may be withdrawn. Allsuch preparations should preferably be rendered sterile.

For injections, the compound may be presented in the form of a sterilesuspension containing up to 20% finely dispersed material in anacceptable medium. Preferably the formulation contains 10% dispersedu-ethoxyethylglyoxal dithiosemicarbazone at an average particle sizebelow 10 ,u.II1., conveniently around 5 m which has an advantageousstorage stability viscosity in an aqueous carrier. Other systems, suchas 10% suspensions in propylene glycol or isopropylmyristate or 5%solution in dimethyl sulphoxide have also been tested with success.

The following examples illustrate the invention.

EXAMPLE 1 (1) Experimental animals (a) Calves.The 74 calves used werehigh grade, predominantly Ayrshire or Friesian, purchased at 1 to 3weeks of age. These were splenectomised 2 to 3 weeks later and infectedwithin 2 weeks after splenectomy. At that time they were all free ofblood parasites and weighed 30 to 52 kg.

(b) Steers.Twenty-nine Guernsey and Friesian high grade steers, negativeto the A. marginale complement fixation test (Peterson, Christensen,Henderson, King, Oglesby, Poelma, Ruby and Willers, Proc. 61st AnnualMeeting, (1957) were purchased from two farms in Kenya on both of whichrigorous tick control was practised. These steers averaged 3 years ofage (range 27 to 48 months) and weighed 310 to 540 kg.

While held at the laboratory a strict acaricidal regimen was applied,ticks being controlled by spraying at 4 to 5 days intervals withtoxaphene and dioxathion at the manufacturers recommendedconcentrations.

(2) Infective inocula Calves were infected by the intravenous injectionof blood containing 1 to 2 strains of A. marginale.

(a) Sixty-six were inoculated with the Onderstepoort, South African,strain which had been received from Professor W. O. Neitz andsubsequently passaged at this laboratory. An intravenous inoculation of5 ml. blood exhibiting approximately A. marginale parasitaemia (5 to8x10 parasitised erythrocytes) induced an acute anaplasmosis reactionfollowing a 5 to 7 day prepatent period. The infections recorded herewere induced with passages between 110 and 155.

(b) Eight calves were infected with the Nevada, U.S.A., strain of A.marginalc, kindly provided by Dr. K. L. Kuttler. Two each were infectedwith the first 4 passages of this strain following resuscitation from acarrier splenectomised calf (20 ml. carrier blood, and 5 ml. blooddemonstrating 8% and 1% parasitaemia respectively). The resultantprepatent period following infection ranged between 15 and 44 days.

The adult steers were infected with 3 consecutive passages (151 to 153)of the Onderstepoort strain. Each animal received 10 ml. infected bloodcontaining 1.9, 2.2 or 3.O 10 parasitised erythrocytes according to theinoculum used. These represented parasitaemias of 33%, 35% and 76% A.marginale, which induced patent infections following 7 to 12 dayprepatent periods.

(3) Test drug a-ethoxyethylgloxal dithiosemicarbazone was administeredin the following formulations:

(a) as a drench made up in water containing:

(1) micronised powder of mean particle diameter of v 5.8 microns;

(2) dispersible granules of the above powder with a wetting agent;

(b) as an intravenous injection of:

(l) 1 0% or 20% aqueous suspension;

(2) 20% suspension in propylene glycol;

(3) 20% suspension in isopropylmyristate; (The mean particle diameter inthe suspensions was 4.6 microns).

(4) 20% solution in dimethylacetamide and propylene glycol.

(4) Observations Giemsa stained, thin blood films were prepared forestimation of parasitaemia; approximately 1,000 erythrocytes werecounted and the number of parasitised erythrocytes takes to the nearestwhole number percent. Haemoglobin estimations, using the alkalinehaematin method of Clegg and King, Brit. Med. J. (1942), were made witha photoelectric colorimeter in comparison with a Gibson and HarrisonStandard. In calves, peripheral capillary blood samples were used forthese observations but in the steers jugular venous blood, taken intodisodium edetate, was used. These observations were made 3 to 7 times aweek, being most frequent at the time of patent parasitaemia,

and continuing to death or the completion of a relapse. Daily rectaltemperatures were taken throughout the trials.

(5) Trials These trials were intended to evaluate the efficacy of thetest compound in controlling both multiplication of A. marginale and thepathological consequences thereof in the bovine host during the patentparasitaemia rise of an acute anaplasmosis reaction. In each trial theresponse in treated groups was compared with the reactions observed in anumber of untreated controls.

(a) Splenectomised calves.--Groups of 4 to 8 calves were infected. Whenthe percentage parasitised erythrocytes reached levels of 1% to 59% andwere doubling daily, a single treatment was given to calves taken atrandom, leaving 1 or more calves of each group untreated as controls ofinfectivity and virulence. The delay between sampling for haematologicalestimations and treatment was approximately six hours. Forty-two treatedcalves were controlled by 24 untreated calves, using the Onderstepoortstrain and 4 treated calves were controlled by 4 untreated calves usingthe Nevada strain.

(b) Steers-The steers were infected with successive passages of theOnderstepoort strain in groups of 7, 12 and 12 with 2, 5 and 5 untreatedcontrol animals for the respective groups. Treatment was delayed untilclinical anaplasmosis was apparent. The subjects were then showing highparasitaemias (11% to 56%), recordable rises in rectal temperatures andfalls in haemoglobin levels.

(6) Results Results are summarised in the table.

Of 24 untreated control valves infected with the Onderstepoort strain ofA. marginale, results are only given from 20. Three of those excludedwere short to provide infective material for serological antigenproduction when showing 71, 79 and 80% parasitaemia respectively. Thefourth underwent an abnormal, subfatal reaction complicated by thepresence of T heileria mutans and Eperytlzrozoon teganodes. It reached apeak parasitaemia of 64% and a minimum harmoglobin level of 2.2 g./100ml. In the controls whose reactions are summarised in the table, peakparasitaemias ranged between 55% and and minimum haemoglobin levels,generally assessed on the day before death, between 5.4 and 1.8 g./ ml.

In comparison, the treated calves infected with the Onderstepoort strainexperienced less severe reactions. Parasite multiplication and itseffects were suppressed by the test drug in both the oral forms at 50mg./kg. and the 4 formulations given intravenously at 10 mg./kg. Thedevelopment of anaemia was most effectively controlled when treatmentwas initiated early in the patent infection, but if delayed untilparasitaemia was high, blood destruction was not halted. The singledeath in treated calves was that of a calf treated when exhibiting a 59%parasitaemia. Parasite multiplication appeared to have been suppressedeffectively, this being the peak parasitaemia attained, but thehaemoglobin level fell after treatment from 9.8 g./ 100 ml. to 3.2g./100 ml., death being attributed to oligocythaemia.

The anaemia was not apparently directly related to the persistence ofvisible marginal bodies as seen in Giemsa stained blood films. Except inthe one fatal case, parasitaemia was invariably reduced to less than 1%following treatment. The parasites, however, did not disappear from theblood immediately after therapy. It was not uncommon to find abnormallysmall bodies which could be considered as A. marginale in 20% to 30% oferythrocytes for up to 20 days following treatment wtihout any evidenceof concomitant anaemia.

The Nevada strain of A. marginale is less virulent and the differencebetween treated and control groups, though apparent, is less obvious. Asingle oral treatment at 50 mg./kg. was relatively more effectiveagainst this parasite than against the Onderstepoort strain.

Single intravenous doses of 10 mg./kg. and 5 mg./kg., using the 20%aqueous suspension, were highly effective in controlling furthermultiplication of A. marginale in susceptible steers undergoing clinicalanaplasmosis reactions to the Ondcrstepoort strain. With treatmentinitiated at relatively high parasitaemia levels (11% to 56%),multiplication appeared to be arrested immediately. The reactionrecorded in untreated controls, compared with that of splenctromisedcalves, was considerably less severe (mortality and more variable (peakparasitaernias 6% to 77% However, even where relatively lowparasitaernias were observed in control cattle, significant anaemiadeveloped, the haemoglobin level in the steer with a 6% peakparasitaemia falling from a pre-infection level of 10.5 g./l ml. to alow of 3.6 g./100 ml.

A feature of the disease in the steers was that the anaemia developedearlier than in splenectomised calves with comparable parasitaemias.

The effectiveness of a-ethoxyethylglyoxal dithiosemicarbazone incontrolling the multiplication of A. marginale was assessed inaccordance with the method recommended for splenectomised calves byMiller, Levy, Torbert and Oglesby, Proc. 89th Ann. Htg., (1952) and forintact cattle by Lotze, Am. J. vet. Res. (1947). The measure of activityagainst the parasite per se was supported by evidence that treatmentalso controlled pathological effects usually associated with themultiplication of the parasite. Survival rate was markedly increased,only 1 death occurring in 63 treated animals, and anaemia waseffectively arrested.

had been moved into a heavily infected area, whilst 8 out of 10 controlcattle previously vaccinated with inactivated or attenuated anaplasmavaccine broke under the challenge and developed the sympotms in the samearea during the same period.

I claim:

1. A method for the treatment of anaplasmosis in cattle, comprising theadministration of an anaplasmosis effective treatment dosage of'u-ethoxyethylglyoxal dithiosemicrabazone to the infected animal.

2. A method according to claim 1 in which the a-ethoxyethylglyoxaldithiosemicarbazone is administered orally or intravenously.

3. A method according to claim 2 wherein the animal is orallyadministered wethoxyethylglyoval dithiosemicarbazone in a dosage of 20mg./kg. of animal bodyweight.

4. A method according to claim 2 wherein the animal is intravenouslyadministered a-ethoxyethylglyoxal dithiosemicarbazone in a dosage of tomg./kg. of animal bodyweight.

5. A method according to claim 4 in which a formulation containing 5 toof finely dispersed a-ethoxyethylglyoxal dithiosemicarbazone in aninjectable liquid medium is intravenously administered.

6. A method according to claim 5 in which the average particle size isabout 5 am.

7. A method of preventing anaplasmosis in cattle comprising theadministration of an anaplasmosis prevention TABLE.ACUTE EXPERIMENTALANAPLASMOSIS. A COMPARISON BETWEEN TREATED AND UNTREAIED ANIMALSTreatment with a-ethoxyethylglyoxal dithiosemicarbazone A. marginaleReaction/response, Inleetion: parasitaemia l haemoglobin g./100 ml. .A.marginale Dose No. of M or- Animal type strain Formulation rug/kg. Routeanimals Day T 2 Peak Day 0 3 Day '1 Min. tahty Splenectomised calf.-.OndcrstepoorL" Micronised powder l x 50 p./o. 8 19 11.7 10. 3 7. 2 0/8 0..d0 Dispersible granules l x 50 p./o. 3 26 10. 3 1 1 7. 5 0/3 doAqueous suspension 1 x 10 i./v. 9 23 23 10.9 9. 7 7. 1 ()/9 D0 .doSuspension in propy- 1 x 10 i./v. 9 17 21 10. 3 8.7 6. 2 0/9 leneglycol.D0 do Suspension in isopro- 1 x 10 l./v. 3 6 11 8. 9 9. 3 6. 9 0/3pylmyl'istate. DO .-d0 Solution in dimethyl- 1 x 10 i./v. 10 20 24 11. 810. 3 7. 2 1/ 10 acetamide and propylene glycol. D0 "do Untreatedcontrols 20 82 10.8 3. 1 18/20 Spleneetomised ealf Nevada Mieronisedpowder 1 x p./o. 4 9 9 11.2 8. 9 7. 5 0/ D ..do Untreated controls 4 2110. 8 4. 6 -0/4 Steer Onderstepoort... Aqueous suspension 1 x 10 i./v.10 21 22 10.0 7. 5 6. 7 0/10 o d0 0 1 x 5 i./v. 7 26 26 10. 8 7. 0 4 0/7D0 .(lo Untreated controls 12 43 10. 3 3. 2 5/12 1 Percentageerythrocytes parasitised. 2 Day of treatment. 3 Day of infection.

EXAMPLE 2 Two splenectomised calves were fed a-ethoxyethylglyoxaldithiosemicarbazone daily at a dosage rate of 1 mg./kg. for 10 days. Onthe fifth day they were inoculated with 1.0 ml. anaplasma carrier blood.

Forty-seven days after inoculation the animals showed no prasitaemia.

EXAMPLE 3 Three adult anaplasma carrier Hereford cows were given adosage of 1 mg./kg. u-ethoxyethylglyoxal dithiosemicarbazone orally. Thetreated animals were then shown to be negative as compared with thecontrol animals still remaining carriers.

EXAMPLE 4 5 cattle were infected with A. marginale. After thedevelopment of parasitaemia they were given a single dosage of 10mg./kg. u-ethoxyethylglyoxal dithiosernicarbazone intravenously, andheld in an anaplasmosisfree area for one month.

These animals showed no clinical evidence of anaplasmosis during a fivemonth observation period after they amount of a-ethoxyethylglyoxaldithiosemicarbazone to cattle.

8. A method of preventing anaplasmosis in cattle, comprising a repeatedintramuscular, subcutaneous or oral administration ofa-ethoxyethylglyoxal dithiosemicarbazone at a dosage of about 1 mg./kg.per day to the animal for 10 to 30 days.

References Cited UNITED STATES PATENTS 3,265,570 8/1966 Michaels 424-323JEROME D. GOLDBERG, Primary Examiner

